Subcellular Mechanisms in Pathogenesis of Pulmonary Arterial Hypertension

描述(申请人提供)：特发性肺动脉高压(PAH)是一种持续进展性疾病，其特征是内皮细胞和血管内皮细胞的增殖、增大和空泡化抵抗细胞凋亡。在对PAH发病机制的讨论中，特别遗漏了对亚细胞膜以及质膜和细胞内部之间的蛋白质/受体运输机制的考虑。我们将测试一种假设，即在这种疾病中，血管相关蛋白的整个篮子的变化(包括各种受体、信号通路分子和eNOS水平的增加/减少)在很大程度上是由于由于高尔基体水平的细胞内运输缺陷而导致这些蛋白错误地定位到错误的亚细胞间隔(“高尔基阻断假说”)。我们认为新的参与者(各自的高尔基系绳、圈套和1-SNAP和NSF)和旧参与者(错位的eNOS和亚细胞内NO减少，突变的BMPRII物种)都参与了“引起”PAH的亚细胞机制。目的以Giantin、GM130、p115、GS28和1-SNAP为研究对象，对原代培养的HPAECs和HPASMCs在不同PAH模拟条件下高尔基体形态、放大和碎裂的高分辨率三维成像研究。AIM II将从eNOS亚细胞错位的角度处理这些变化的后果。这些研究将在(1)暴露于无清除、低氧或MCTP的HPAECs培养中进行，(2)在经MCT处理的大鼠、人PAH和感染Shiv-nef的猕猴肺血管病变的福尔马林固定切片中的细胞中进行。目的验证PAH疾病相关的BMPRII突变种可使eNOS和转运中介蛋白错位定位于HPAECs和HPSMCs，从而对细胞内转运产生显性负效应的假说。综上所述，拟议的研究代表了在PAH中检验高尔基封锁假说的下一步。该项目完成后，我们将在细胞培养和组织切片中阐明高尔基体形态的变化，获得eNOS和1-SNAP亚细胞错误定位的证据，并为BMPRII突变如何扰乱细胞内运输提供新的机制，最终在PAH病变的肺血管细胞中观察到细胞表型。 公共卫生相关性：在美国，每年有15,000名患者死于肺动脉高压(PAH)。目前尚不清楚是什么原因导致了PAH。尽管BMPR2基因的突变已被证实，但经过10年的研究，其机制仍未阐明。我们提出了一种新的方法来看待这种疾病的发病机制，从整体上扰乱了肺动脉内皮细胞和平滑肌细胞的细胞内运输，导致血管相关蛋白在细胞表面和细胞内的位置错误地放置。

DESCRIPTION (provided by applicant): Idiopathic pulmonary arterial hypertension (PAH) is an unrelentingly progressive disease characterized by proliferative, enlarged, and vacuolated endothelial and smooth muscle cells resistant to apoptosis. Singularly missing from discussions of the pathogenesis of PAH is consideration of subcellular membrane- and protein/receptor-trafficking mechanisms between the plasma membrane and the cell interior. We will test the hypothesis that the entire basket of alterations in vasorelevant proteins (including increases/decreases in levels of various receptors, signaling pathway molecules and eNOS) in this disease is largely due to mislocalization of these proteins to the wrong subcellular compartments due to defects in intracellular trafficking at the level of the Golgi apparatus ("Golgi blockade hypothesis"). We implicate both new players (respective Golgi tethers, SNAREs and 1-SNAP and NSF) and old players (mislocalized eNOS and reduced subcellular NO, mutant BMPRII species) in the subcellular mechanisms that "cause" PAH. Aim I will deal with high-resolution 3-D imaging studies of the alterations of the Golgi apparatus morphology, enlargement and fragmentation under various PAH-mimetic conditions [NO scavenging, hypoxia and monocrotaline pyrolle (MCTP)] in cultures of primary HPAECs and HPASMCs with a focus on giantin, GM130, p115, GS28 and 1-SNAP. Aim II will deal with the consequences of these changes in terms of the subcellular mislocalization of eNOS. The studies will be carried out in (1) cultures of HPAECs exposed to NO-scavenging, hypoxia or MCTP, and (2) in cells in formalin-fixed sections of vascular lesions in lungs derived from the MCT-treated rat, human PAH and the SHIV-nef-infected macaque with pulmonary vasculopathies. Aim III will test the hypothesis that PAH-disease-associated BMPRII mutant species can mislocalize eNOS and trafficking-mediator proteins in HPAECs and HPSMCs causing a dominant-negative effect on intracellular trafficking. Taken together, the proposed studies represent the next step in testing the Golgi blockade hypothesis in PAH. Upon completion of this project we will have elucidated changes in Golgi apparatus morphology in high-resolution 3-D in both cell culture and in cells in tissue sections in this disease, obtained evidence for subcellular mislocalization of eNOS and 1-SNAP, and provide a novel mechanism for how mutations in BMPRII might disrupt intracellular trafficking culminating in the cellular phenotype observed in pulmonary vascular cells in PAH lesions. PUBLIC HEALTH RELEVANCE: Fifteen thousand patients die of pulmonary arterial hypertension (PAH) every year in the U.S. It is not clearly understood what causes PAH. Although mutations in the BMPR2 gene have been implicated, the mechanisms have not been elucidated despite 10 years' of investigations. We propose a novel way of looking at the pathogenesis of this disease in terms of a global disruption of intracellular trafficking in pulmonary arterial endothelial and smooth muscle cells which result in the incorrect placement of vasorelevant proteins on the cell surface and in locations within the cells.